Abstract: Homozygosity for a single mutation (HFE C282Y) is responsible for the vast majority of cases of hemochromatosis, yet phenotypic expression in homozygotes varies widely. The hypothesis to be tested in this amended competing renewal is that modifier genes are responsible for the phenotypic variability. To identify modifier genes, we propose three specific aims. Specific Aim 1: Identify candidate modifier genes on chromosomes affecting iron absorption. Iron metabolism in inbred mice varies in a strain-specific manner. The A/J strain is a "high iron" mouse and absorbs more iron and has higher liver iron content compared to the "low iron" C57BL/6J mouse. During the previous funding period, we phenotyped 21 chromosome substitution strains (CSS) that represent the 21 mouse chromosomes of A/J on an otherwise C57BL/6J genetic background. We used Recombinant Cogenic Strains (RCS) to localize a modifier region on the distal end of chromosome 5. Stratification of candidate genes will be based on tissue-specific expression differences between A/J, C57BL/6J and specific recombinant congenic strains. Specific Aim 2: Characterize the regulation of iron uptake, transport and export by the absorptive enterocyte. Preliminary results indicate that in the absence of an erythropoietic, iron stores, or hepcidin-mediated signal, the amount of iron mice absorb is regulated by a mechanism intrinsic to the enterocyte. We will identify genes that are differentially expressed in enterocytes during the response to altered dietary iron content. These regulated genes will become candidate modifier genes. Using this strategy we have identified at least two candidate modifier genes, transferrin receptor 2 and a member of the chloride transporter family. Specific Aim 3: Characterize candidate modifier genes that affect the iron phenotype in Hfe -/- mice and the homologous human genes in patients with hemochromatosis. Candidate modifier genes in the mouse will be sequenced, cloned, expressed in vitro and antibodies generated against the gene products. Western analysis will be used to confirm gene expression differences detected in Specific Aims 1 and 2. Candidate gene sequences will be determined in other high iron and low iron strains. A unique collection of human hemochromatosis pedigrees will be utilized to compare the sequences of homologous candidate modifier genes in sib pairs with concordant and discordant clinical phenotypes. Common alleles should be found among concordant sibs and not among discordant sibs. The function of candidate genes will be tested using cell culture models of iron transport and iron homeostasis. Identification of modifier genes may provide a method for risk assessment for hemochromatosis and possible therapeutic strategies.